Our group focuses on the discovery, biosynthesis, structure, and function of natural products (NPs), which rep- resent the largest source of chemical matter for small molecule drugs. The most prolific producers of medicinally relevant NPs derive from a group of bacteria known as the actinomycetes. Retrospective genome analysis has repeatedly shown that synthetically talented microbes, including actinomycetes, are capable of synthesizing many more NPs than are currently known. Exploring the undiscovered majority, this so-called NP ?dark matter?, holds enormous potential for not only improving human health by expanding our pharmaceutical repertoire but also for advancing the fundamental study of biology given the critical role of NPs as chemical probes. This proposal outlines a chemistry-centric NP discovery strategy designed specifically to address tradi- tional shortcomings. Despite the success of traditional NP discovery methods, the most prevalent, bioassay- guided isolation, suffers from inherent biases that leads to unacceptably high rates of rediscovery. Rather than track a NP by its bioactivity, we have elected to identify NPs by the presence of organic functional groups that can be chemoselectively derivatized without the need for purification. A review of the 250,000 NPs present in the Dictionary of NPs shows that ~50% contain such a functional group. We will detect these groups with pmol sensitivity using mass spectrometry in the context of crude bacterial extracts after reaction with a suitable probe. Importantly, the presence of the functional groups we are targeting can be bioinformatically predicted based on known biosynthetic pathways. We have termed this NP discovery procedure reactivity-based screening (RBS) and have recently reported the discovery of thiopeptides and polyketides by nucleophilic 1,4-addition as well as non-ribosomal peptides (NRPs) by oxime ligation. A custom bioinformatics software program has also been developed by our group to aid in predicting the presence of various targetable organic functional groups. Employing our in-house collection of ~10,000 actinomycetes, many from rare, understudied taxa, we have de- vised this project to consist of three independent but interconnected aims. For Aim I, the focus is on novel thiopeptides, which are extensively posttranslationally modified peptides best known for their ability to block bacterial protein translation. Aim II focuses on the discovery and characterization of novel polyketides while Aim III is directed towards NRPs. Each aim will elucidate NP structure and bioactivity using a multi-tiered strategy. The latter two aims also introduce new probe chemistry beyond the reactions already mentioned. The central hypothesis of this project, for which we have a large amount of supportive data, is that NP discovery can be accelerated through the marriage of genomics prioritization and RBS. We envision this approach to be highly enabling and increase in power as more genomes appear in public databases.